| OBJECTIVEDiarrheal diseases caused by bacterial pathogens are a major problem worldwide, especially in developing countries. The species Escherichia coli comprise intestinal and extraintestinal pathogens. The intestinal pathogens are also known as diarrheagenic E. coli (DEC) of which six categories have been characterized:enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffusely adhering E. coli (DAEC). Diarrheal diseases due to the different DEC are an important public health problem in many parts of the world. The aims of this study are to evaluate the prevalence,categories,serotypes,distribution of virulence factor genes of DEC isolated from acute diarrheal cases in Zhejiang region by polymerase chain reaction (PCR) method targeting 12 virulence factor genes and its relationship with age, sex, season, regional distribution.We also sought to determine the relations of serotypes and virulence factor genes of the isolated strains and whether it is feasible to identify and classify isolates of diarrheagenic E. coli on the basis of major serotypes.METHODSEleven targeted virulence-associated genes bfpB. escV,escV,stx2,elt,estla, estlb,invE. pic. aggR and astA ere selected to molecular detect simultaneously five categories of diarrheagenic E. coli (EPEC,EHEC,ETEC,EIEC,EAEC) by multiplex polymerase chain reaction and the daaD was chosen to detect DAEC by single polymerase chain reactions. Stool samples were collected from 1500 patients with diarrhea in five cities(Hangzhou,Shaoxing,Ningbo,Lishui,Quzhou) of Zhejiang Province, China, from June 2009 to August 2010. The specimens were cultured for E. coli and other pathogens by conventional cultivation methods, E. coli strains were screened for DEC by multiplex PCR and single PCR method. The O and H serotyping method were used to identify the E coli isolates that recognized DEC. The rate of isolation of DEC and other organisms was compared among individuals in different age,sex,season, regional distribution. Analysis of the major serotypes. virulence genes and their correlation is performed. The coincidence rate is compared of bacteria colony mPCR with stool specimens directly mPCR method for diagnosis DEC from 100 clinical specimens.RESULTS1. Among 401(26.7%) diarrheal etiologic agents isolated from 1500 diarrheal cases in five cities of Zhejiang Province,191 (12.7%) corresponded to diarrheagenic E. coli indicating a high prevalence among the diarrheagenic population. EAEC and ETEC pathotypes were the most prevalent.Of the 191 diarrheagenic E. coli detected by mPCR,122 (63.8%) were EAEC,46 (24.1%) were ETEC,15 (7.9%) were EPEC,7 were EHEC and 1 (0.5%) was EIEC, No DAEC strains were identified in the specimens.2. The incidence of DEC and ETEC in the adult group (14.6%,4.3%) was higher than that of in the infant group (11.0%,1.3%) and in the children group (4.5%,1.1%). The EPEC, EHEC, EIEC, EAEC was no significant difference among the three groups.3. Among 191 DEC isolated from 5 regions,DEC, EAEC, ETEC have regional differences, the detection rate of Shaoxing are higher than that of Hangzhou, Hangzhou are higher than that of other areas, however, EPEC, STEC, EIEC have no regional differences.4.The prevalence of DEC and pathotypes between 789 male patients and 711 female patients with acute diarrhea was not statistically significant.5. The incidence of DEC in 1500 cases of acute patients have some differences in different seasons in a year,the high detection rate of DEC and ETEC in summer and autumn (13.7%,3.6% respectively) in contrast to that of in winter and spring (8.8%, 0.7% respectively),There was a significant difference between the two different seasons (p=0.036,0.014), and EPEC, STEC, EIEC, and EAEC was no significant difference.6. Of the 191 DEC,65 (34.0%) were serotypable, belonging to 21 species serotype O. The most prevalent serogroups among these isolates were 06 (15,23.1%), 025 (9,13.8%) and 0169 (6,9.2%). These three serotypes are responsible for the majority of this area. There were 4 different EPEC serotypes and 1 STEC serotypes 0157:H7. The major ETEC serotype was 06,025,0159. EAEC serotypes belonging to 17 species, mainly serotypes was 06,015,0169. no submit to EIEC serotypes.7. A total of 10 virulence-related gene were detected in 191 DEC expressing 18 patterns in which most prevalent genetype model was astA (90 strains,47.1%), estla+ estlb (31 strains,16.2%), elt (9 strains,4.7%);In 65 serotyped strains,29 strains (44.6%) carried astA,17 strains (26.2%) carried estla+estlb,6 strains (9.2%) carried elt; The first 3 serotypes 06 to carry the major genotype estla+estlb and astA,025 major genotype elt,0169 major genotype astA; 126 untyped strains,61 (48.4%) harboring astA,14 strains (11.1%) harboring estla+eslb.Several different combinations of the virulence markers were found among the EAEC isolates. The most prevalent combination was astA-pic.8. The results colony and stool specimens of genomic DNA PCR determination of 100 clinical specimens DEC showed that there was 89% correlation with culture isolate-based and direct stool PCR result.12 cases were both methods positive and consistent,77 cases were both negative,11 cases were inconsistent.CONCLUSION1. Our study demonstrated high prevalence (12.7%) of DEC in Zhejiang area and DEC is the main pathogen of acute diarrhea.There are at least five groups of DEC in this area, the most prevalent is EAEC, followed by ETEC, EPEC. EHEC, EIEC are uncommon, DAEC is rare. This is the first report on the molecular characterization of E. coli in this geographic area.2. The incidence of DEC and ETEC in adult is higher than that of in low age group, While DEC,EAEC ETEC in Shaoxing and Hangzhou are frequently detected; The prevalence of DEC and ETEC in summer and autumn is higher than that of in winter and spring;howere,there is not any statistically difference is observed between male and female patients with acute diarrhea.3. There is higher serotypable rate (34.0%) in this area and at least 21 O serogroups are obtained, mainly serotype maybe 06,025,0169. it is suggested that serogrouping-based methods are inadequate for the identification of DEC isolates, although they are useful for the identification of a limited number of serogroups.4. Our study also recognized the diversity of E. coli virulence-related gene in these area by detecting clinical isolates,genetype patterns for both serotyped and untyped isolates were very similar, with astA,estla+ estlb-elt are the most prevalent genetype model. According to our data, there are particularly high virulence gene carriage rates in some of the commercially defined 0 serogroups.06,0169 serogroup inclination to carry astA while 025 serotype carrying elt is more commonly.5. In comparison to the mPCR assay for diagnosis DEC after culturing the bacteria, the mPCR assay starting directly from extraction of stool DNA has been found to be simple and less time-consuming.The latter can be used in emergency situations such as a diarrhea outbreak. |
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